Protein translocation in the rough endoplasmic reticulum (RER) represents the point of entry of secretory and membrane protein precursors into the cellular protein trafficking pathways and comprises both the transfer of secretory precursors from the cytoplasm to the RER lumen and the assembly of membrane protein precursors into the RER bilayer. Protein translocation in the RER requires protein factors acting on the cis-, or cytoplasmic, and trans-, or lumenal, sides of the RER membrane. Cis-side factors target precursors to the RER membrane and maintain the protein in an unstructured state. The function of trans-side proteins in translocation has not been defined and is the focus of the proposed studies. In addition to our interest in translocation as an essential cellular process, recent studies on the assembly and sorting of membrane protein precursors indicate a pressing health interest in the elucidation of the mechanism of translocation. In the case of the visual pigment rhodopsin, for example, defects in assembly of various rhodopsin mutants in the RER have been demonstrated to result in autosomal dominant retinitis pigmentosa, a progressive blinding disease. The experimental analyses will exploit a well-characterized in vitro cell-free system which accurately mimics in vivo protein translocation. The role of lumenal proteins in translocation will be assessed using a series of reporter proteins; the secretory precursor, preprolactin, the type I integral membrane protein VSV-G (vesicular stomatitis viral glycoprotein), the type II integral membrane protein HNA (hemagglutinin/neuraminidase protein of Newcastle virus) and the polytopic membrane protein opsin, in experiments involving the depletion and biochemical reconstitution of wild-type and mutant forms of the lumenal proteins in isolated RER derived microsomal vesicles. Chemical cross-linking studies will be performed to identify associations of lumenal proteins with precursors at different stages of translocation. These studies will be elaborated in experiments designed to determine the mechanism of lumenal protein function and will include studies on the role of domain specific interactions between precursors and individual lumenal proteins. Such studies will allow characterization of the structural determinants of the precursor necessary for efficient translocation in the RER and further the mechanistic description of protein translocation.